KR101773832B1 - Pyralid moths eggs, its producing method, and method for producing recombinant protein by using pyralid moths eggs - Google Patents

Abstract

The present invention relates to a means and a method for increasing expression efficiency of recombinant proteins and, more specifically, to a means and a method for optimizing industrial production of recombinant proteins from eggs of Pyralid moths, especially eggs of Ephestia kuehniella. Furthermore, the present invention relates to a device for performing the method of the present invention with transformation or transduction and transfection by not only eggs of Pyralid moths including recombinant baculovirus but infected eggs of Pyralid moths of recombinant baculovirus, recombinant baculovirus or bacmids.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for producing a recombinant protein, and a method for producing the recombinant protein using the same,

The present invention can be included in the field of biotechnology and is intended to increase the efficiency of recombinant protein expression. More specifically, the present invention relates to a method for improving the efficiency of recombinant protein expression, Lt; RTI ID = 0.0 > of recombinant < / RTI > proteins in eggs. Furthermore, the present invention relates to a method for transforming or transducing recombinant baculovirus with recombinant baculovirus, recombinant baculovirus, recombinant baculovirus, or recombinant baculovirus, recombinant baculovirus or bacmids, transduction or transfection.

Recombinant protein expression systems used as vaccines, therapeutic molecules or diagnostic reagents utilize a variety of expression vectors to generate bacterial (prokaryotic) Escherichia coli and eukaryotes (Yeast) have been developed based on bioreactors such as plant cell culture technology, animal cell culture technology, and insect cell culture technology.

In particular, since the baculovirus expression vector system (BEVS) using insect cells developed from the 1980s has a high possibility of over-expression and a rapid development speed, it is difficult to produce all-use recombinant proteins It is known as the most efficient method for. Currently, recombinant baculovirus vectors, most commonly used in industry for expression of recombinant proteins, are suitable hosts for expression hosts such as Spodoptera frugiperda 9 (Sf9) or 21 (Sf21) and autograpa californica nuclear polyhedrosis virus (AcMNPV), and since the development of BEVS, hundreds of recombinant proteins from cytosolic enzymes to membrane-bound proteins through recombinant baculovirus-infected insect cells Was successfully produced.

However, a recombinant protein expression system based on a stainless steel stirred-tank bioreactor requires years of time and high cost to initial construction, requires a highly skilled workforce to operate the system, is susceptible to contamination, There is a problem of low reliability because it is difficult to verify the result. In the scale-up production phase from the 1990s, the above problem can be solved somewhat by using a wave bioreactor using a disposable plastic bag, but in the final production stage, inefficiency, high cost, technical complexity, Faced with limited scalability problems, it becomes a technical and economic barrier to producing new or existing recombinant proteins.

Recently, Ebola Virus Disease (EVD), which is a mixture of three kinds of monoclonal antibodies, has been applied to a living tobacco (Nicotiana tabacum) plant rather than bioreactors based plant cell culture technology Generally produced by transfection but not for production of recombinant proteins due to the relatively low productivity and production cycle problems of tobacco which takes several months. Insects, however, can grow up to about 5000 times in size within two weeks because of their rapid metabolism. In mammals, a single cell can produce up to about 50 μg of protein per day compared to insect bombycidae, Is known to be the most efficient protein producer because one silk gland cell of Bombyx mori (Silkworm) can produce about 80 ug of protein per day. Therefore, as living biofactories, insects are promising alternatives to conventional bioreactor-based microbial and cell culture technologies due to the flexibility, scalability, automation potential, efficiency of development and rapid development of mass production, Also known as bioreactor using live insects.

Larvae and pupae of Lepidoptera such as Trichoplusia ni (Cabbage looper) are the most commonly used insect bioreactor, and AcMNPV or silkworm nuclei Bombyx mori nuclear polyhedrosis virus (BmNPV) vectors. The insect larvae and the pupae body fluids are aseptic and contain protease inhibitors (Pls) so that the proteolytic degradation is not likely to occur and thus the substances expressed by BEVS can be stably present. In the case of the cell culture method, the cell culture medium contains fetal bovine serum (FBS), and it is necessary to use a complicated biochemical purification method in order to separate the target recombinant protein, but it is possible to recover relatively easily in insect body fluids. In particular, AcMNPV and BmNPV are capable of expressing foreign genes in almost all cells except for some tissues in the trunk, and their recombinant protein productivity is remarkably higher than that of cell culture methods. In particular, according to KR10-0921812, in the Sf9 insect cell culture method, the cellular enzyme activity showed an expression efficiency of 15.25 units (U) per 1 ml, whereas the expression efficiency was about 226 times as 3439 units per 1 ml in the silkworm larvae It looked.

Methods for inoculation of recombinant baculoviruses against insects used as insect bioreactors include a method in which recombinant baculovirus is directly injected into a larva or a pupa by a syringe, a method in which recombinant baculovirus A method of oral inoculation which infects the mucosa through the gastrointestinal mucous membrane by feeding the mucosal powder into the gastrointestinal mucosa and the respiratory organs by immersing the larva or the pupa in the liquid containing the recombinant baculovirus, (soaking) method are known. Comparisons in silkworm moths have shown that most proteins have the highest expression rates in larvae than in pupae, and that the susceptibility to recombinant beculovirus infection decreases as the age of silkworm moth pupa increases. Also, since the silkworm moth pupa can not use the oral inoculation method, it is tedious and time consuming to perform the injection method by hand, and the thick cocoon covered in the pupa before the recombinant baculovirus should also be manually removed. Due to the general reduction of protein expression and difficulty in manipulation of the silkworm moth pupa, insect bioreactors are generally used in the scale up stage rather than in the mass production stage. In addition to the injection method or immersion method for larvae and pupa, Lt; / RTI >

Recently, in WO2012 / 168493, WO2012 / 168492 and WO2017 / 046415, a new recombinant baculovirus vector, a cabbage-chestnut pupa, and a pupa of about 3,000 horses per hour were used to continuously introduce recombinant baculovirus An automated system has been described. However, the larvae of the silkworm moth and moths, Saturniidae, and Sphingidae, which have been mainly used as insect bioreactors to date, feed on the fresh leaves of specialized host plants, respectively, Artificial feeds that are not cheap are also used. In addition, due to the relatively large size of the larvae, there is a problem of insufficient breeding space and excessive labor force input for the breeding of animals when the number of birds increases, and the pupa is covered with a thick cocoon. Due to all these drawbacks, silkworms and moths, moths moths, and lepidopteran insects belonging to the mushrooms are not suitable for industrial mass rearing, and recombinant protein expression systems using them are mainly used only in the scale up production stage. In order to solve the problems of inefficiency, high cost, technical complexity and limited scalability by completely replacing the stainless steel agitation tank bioreactor until the final stage of mass production, a new insect bioreactor suitable for industrial mass rearing was selected and recombinant baculovirus A new recombinant protein expression system that can automate the inoculation of recombinant proteins is needed.

In the insect industry all over the world, large quantities of pyralid moths are added to the stored foods for the production of commercial natural enemies, It is widely used as an alternative host of parasites such as Trichogramma spp. And substitute diets of predatory predators such as Orius spp. . In particular, Cadra cautella (Almond moth) and Mediterranean flour moth (Ephestia kuehniella) are the most common byproducts such as rice bran from wheat rice processing and wheat bran from wheat processing , The material cost is low, and the size of the larvae is about 10 ~ 24㎜ and there is no cannibalism. In addition, since there are many eggs scattered from about 100 to 700 eggs per female, and the egg has a size of about 0.5-1.0 mm long and scattered individually, It is possible to harvest the larvae in large quantities by simply separating them.

However, all insect eggs are surrounded by an egg shell with a multilayered structure of vitelline membrane, waxy layer and chorionic layer outside the cell membrane, through which breathing is possible It prevents infection of viruses and bacteria from the outside. As a result, it has been used in large quantities in the world for mung bean sprouts which are used for storage foods such as Mediterranean mushroom moths. However, since it is very difficult to inoculate the recombinant baculovirus, it has been used as an insect bioreactor for the expression of recombinant proteins not. According to WO2010 / 081078, 90% of D-limonene + 5% of cocamide deia is used for observation of embryogenesis process in Drosophila spp. (Embryo permeabilization solvent (EPS) and sodium hypochlorite (EPS) consisting of 5% Cocamide DEA + 5% ethoxylated alcohol were used to remove the chick embryo and wax layer Desc / Clms Page number 2 > However, insect bio-bioreactors, which are suitable for large-scale rearing and almost completely automated inoculation of recombinant baculoviruses to reduce costs associated with recombinant protein expression and increase efficiency from the scale-up production stage to the final production stage on an industrial scale Have not yet been reported.

Literature 1 US5472858 (Wisconsin Alumni Research Foundation) 1991. 06. 04. [Document 2] KR10-0426462 (Republic of Korea) 2001. 02. 16. [Reference 3] KR10-0921812 (Dong-A University Industry-Academic Cooperation Group) 2006.11.11. [Document 4] KR10-1581184 (Alternative Gene Expression S .L. (Ergenex), Inst. Tetonacilonide Technolopia Agropecaria (Intra)) 2007. 07. 27. [Document 5] KR10-2011-0081810 (Alternative Gene Expression S.L. (Ersenex)) 2008. 09. 02. [Reference 6] KR10-1053217 (Korean Insect Insect Research Institute, Inc., Gyeonggi-do) 2008. 11. 28. [Patent Document 7] WO2010 / 081078 (The University of Vermont, State Agriculture College) 2009. 01. 09. [Patent Document 8] US2011 / 0314562 (Executive Yuan, Miaoli (TW); Miaoli District Agricultural Research, Extension Station, Council of Agriculture) [Reference 9] KR10-1563583 (Chungbuk National University Industry-University Collaboration Foundation) 2011. 05. 17. [Patent Document 10] EP 2858489 (Alternative Gene Expression, S. L.) [Literature 11] EP 285 849 (Alternative Gene Expression, S. L.) [Document 12] US9303251 (Sysmex Corporation) 2013. 03. 29. [Literature 13] WO2017 / 046415 (Alternative Gene Expression, S. L.) 2015. 09. 17.

 [Document 1] Biocontrol Science, Technology. Effect of different cold storage periods on parasitization performance of Trichogramma cacoeciae (Hymenoptera, Trichogrammatidae) on eggs of Ephestia kuehniella (Lepidoptera, Pyralidae): Nihal, 2004, http://dx.doi.org/10.1080/09583150410001683529, Volume 14 Issue 5 , Pp. 441-447  [Literature 2] Entomological Society of America. New cell lines from Ephestia kuehniella: characterization, susceptibility to baculoviruses: Dwight and Ferkovich, 2004, http://www.bioone.org/doi/full/10.1673/031.004.0901, Journal of Insect Science. Volume 4 (9), page 1-5.  [Literature 3] Current Pharmaceutical Biotechnology. Silkworm as a Host of Baculovirus Expression: Usami, Suzuki and Nagaya and Kaki and Ishiyama, 2010, https://doi.org/10.2174/138920110791112013, Volume 11, 246-250  [Document 4] Short Communication. A Review of the McMorran Diet for Rearing Lepidoptera Species with Additions Further Species: Hervet and Laird and Floate, 2016, https://academic.oup.com/jinsectscience/article-lookup/doi/10.1093/jisesa/iev151, Journal of Insect Science, Volume 16, page 17

The present invention has been proposed in order to solve the above-mentioned problems of the previously proposed methods, and it has been proposed to combine recombinant baculovirus vectors derived from AcMNPV for the production of recombinant proteins used for diagnosis, vaccination, The chrysanthemum shell and the wax layer of egg shell are removed using nontoxic chemicals such as an embryo permeation solution and sodium hypochlorite, or a composition thereof, so that a recombinant baculovirus It is an object of the present invention to provide an insect bioreactor which can be completely automated through a simple inoculation method in which viruses are sprayed or dipped in a liquid containing recombinant baculovirus.

The present invention relates to means and methods for increasing the efficiency of recombinant protein expression, and more particularly, to optimizing the industrial production of recombinant proteins in egg shells, especially Ephestia kuehniella eggs, And to a method and a method for doing so. Furthermore, the present invention relates to a method of transforming or transfecting a recombinant baculovirus with recombinant baculovirus, recombinant baculovirus or a recombinant baculovirus or recombinant baculovirus, Lt; RTI ID = 0.0 > a < / RTI >

The recombinant protein of the present invention is preferably a subunit monomeric vaccine, a subunit multimeric vaccine, a virus like particle, a therapeutic protein, an antibody (eg, antibodies, enzymes, cytokines, blood clotting factors, anticoagulants, receptors, hormones, diagnostic protein reagents and green fluorescence ≪ / RTI > protein (GFP; green fluorescent protein).

In the present invention, Pyralis farinalis, Paralipsa gularis, Plodia interpunctella, Aglossa dimidiatus, Corcyra cephalonica, Ephestia kuehniella, (Pyralid moths), which are selected from the group consisting of Ephestia elutella, Cadra cautella, and the like. And it is preferable to mass-produce and supply mushroom eggs belonging to the medaka powdery moth species.

Then, the egg shell surrounded by egg shells having a multilayered structure of a vitelline membrane, a waxy layer and a chorionic layer is treated with a nontoxic chemical to remove the outermost chorion, , Preferably 3% sodium hypochlorite, to remove the chorion. In addition, the chick embryo-free flounder is treated with a non-toxic composition to remove the wax layer, preferably 90% D-limonene + 5% Cocamide DEA + 5% ethoxy The wax layer is removed by treatment with an embryo permeabilizing solution (EPS) composed of ethoxylated alcohol.

After the chick embryo and wax layer of egg shell are removed and the surface disinfected egg shell is sterilized, it is packed in a sterilized container which prevents contamination from the outside but has a ventilation structure for breathing and exchange of internal gas. And preferably in a medicinal flour moth species, the chrysanthemum and wax layer of egg shells are removed and stored in a sterilized container having a ventilation structure at a low temperature of about 4 to 8 ° C. At low temperatures of about 4 to 8 ° C, it can be stored for up to about 4 weeks, so it can be supplied to the whole world through refrigeration transportation using an aircraft.

Then, the recombinant baculovirus originating from Autographa californica nuclear polyhedrosis virus (AcMNPV) is sprayed onto the above-mentioned birds or preferably the above-mentioned birds are immersed in a liquid containing recombinant baculovirus derived from AcMNPV Lt; RTI ID = 0.0 > AcMNPV < / RTI > Also incubated with recombinant baculovirus from AcMNPV for a time sufficient to express at least one recombinant protein.

And incubated for a time sufficient for the recombinant protein to be expressed to recover the protozoan egg containing one or more recombinant proteins and to be stored frozen at -20 캜. Under the above -20 ° C freezing condition, the moths can be stored for up to about one year, and thus it is possible to supply them to the whole world through the refrigeration transportation process using airplanes and ships.

Finally, the frozen egg containing the at least one recombinant protein is crushed. Finally, the at least one recombinant protein is isolated and purified from the liquid in which the flounder is pulverized to produce a final recombinant protein product.

The recombinant protein expression system based on a new insect bioreactor suitable for the industrial mass breeding proposed in the present invention and capable of automating the inoculation of the recombinant baculovirus can be produced by using the existing Baculovirus Expression Vector System (BEVS) and the bioreactor Cost, technical complexity and limited scalability in the production of recombinant proteins through insect cell culture technology based on insect cell culture technology, and the efficiency of recombinant protein expression is improved to 5 times (WO2017 / 046415) to 226 times (KR10-0921812).

It is also possible to replace the wave bioreactor in the scale up production stage as well as the stainless stirred tank bioreactor in the final production stage to reduce costs associated with recombinant protein expression and increase efficiency.

Brief Description of the Drawings Fig. 1 is a diagram showing a whole process of producing a recombinant protein by inoculating a recombinant baculovirus vector derived from AcMNPV to a mungbean egg to which a foodstuff is added according to an embodiment of the present invention. Fig.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings, in order that those skilled in the art can easily carry out the present invention. In the following detailed description of the preferred embodiments of the present invention, a detailed description of known functions and configurations incorporated herein will be omitted when it may make the subject matter of the present invention rather unclear. In the drawings, like reference numerals are used throughout the drawings.

Incidentally, "including" an element means that it may include other elements, not excluding other elements unless specifically stated otherwise.

Brief Description of the Drawings Fig. 1 is a diagram showing a whole process of producing a recombinant protein by inoculating a recombinant baculovirus vector derived from AcMNPV to a protozoan egg according to an embodiment of the present invention. As shown in FIG. 1, the production of recombinant proteins in the protoplast according to the embodiment of the present invention includes a process (1) of removing chick embryo and wax layer of egg shell of eggplant, a method of spraying recombinant baculovirus (2) a step of inoculating the recombinant protein by immersing in water, (2) a step of inoculating the recombinant protein with immersion, and (3) a step of harvesting and purifying the recombinant protein and producing the product in the artificial egg.

The process (1) for removing the chick embryo and the wax layer of egg shells of the present invention is carried out in the order of Pyralis farinalis, Paralipsa gularis, Plodia interpunctella, Aglossa selected foodstuffs selected from the group consisting of Diospyros, Dimidiatus, Corcyra cephalonica, Ephestia kuehniella, Ephestia elutella, and Cadra cautella. (Pyralid moths). The larvae of the myrtle species belonging to the Mediterranean myrtle species are preferably mass produced and supplied.

Then, the egg shell surrounded by egg shells having a multilayered structure of a vitelline membrane, a waxy layer and a chorionic layer is treated with a nontoxic chemical to remove the outermost chorion, , Preferably 3% sodium hypochlorite, to remove the chorion. In addition, the chick embryo-free flounder is treated with a non-toxic composition to remove the wax layer, preferably 90% D-limonene + 5% Cocamide DEA + 5% ethoxy The wax layer is removed by treatment with an embryo permeabilizing solution (EPS) composed of ethoxylated alcohol.

Finally, the chick embryo and the wax layer of egg shell are removed and the surface disinfected egg shell is packed in a sterilized container which prevents contamination from the outside but has a ventilation structure for breathing and exchange of internal gas. And preferably stored in a sterile container having a ventilating structure and stored at a low temperature of about 4 to 8 ° C, preferably in a Mediterranean flour moth species, with the chick embryo and wax layer removed from the egg shell. At low temperatures of about 4 to 8 ° C, it can be stored for up to about 4 weeks, so it can be supplied to the whole world through refrigeration transportation using an aircraft.

The recombinant protein of the present invention is preferably a subunit monomeric vaccine, a subunit multimeric vaccine, a virus like particle, a therapeutic protein, an antibody (eg, antibodies, enzymes, cytokines, blood clotting factors, anticoagulants, receptors, hormones, diagnostic protein reagents and green fluorescence ≪ / RTI > protein (GFP; green fluorescent protein).

In a preferred embodiment, the recombinant protein is a virus-like particle protein, more preferably selected from the following group.

(a) Porcine circovirus capsid protein, preferably porcine circovirus type 2 (e. g., SEQ ID NO: 26),

(b) Foot mouth disease virus VP1, VP3, VP0 protein,

(c) Canine parvovirus VP1, VP2 protein,

(d) porcine parvovirus VP1, VP2 protein,

(e) coat protein of human norovirus genogroup I or II,

(f) Calicivirus coat protein,

(g) human papillomavirus L1 protein, preferably human papilloma virus 16,

(h) Hepatitis E virus protein E2,

(i) Infectious bursal disease virus VP1, VP2, VP3 protein,

(j) Astrovirus ORF2-encoded protein,

(k) Influenza virus HA (e. g., SEQ ID NO: 30), NA, M1 protein,

(l) Hepatitis B Core antigen and surface antigen,

(m) Rabbit calicivirus VP60 protein, preferably rabbit haemorrhagic disease virus RHDVb, RHDVG1 (e.g., SEQ ID NOs .: 32, 33).

(n) human parvovirus VP1, VP2 protein

For example, the recombinant protein may be:

 A porcine circovirus coat protein, preferably a porcine circovirus type 2, e. G., An amino acid sequence of SEQ ID NO: 26, or a nucleic acid sequence of SEQ ID NO: 25 .

 Foot-and-mouth disease virus (FMDV) VP1, VP3, VPO proteins, for example sequences that can be indicated or derived in the following sequence:

  - FMDV serotype O complete genome: GenBank JX570650.1

  - FMDV serotype A complete genome: GenBank HQ832S92.1

  - FMDV serotype C complete genome: GenBank AYS93810.1

  - FMDV serotype SAT 1 complete genome: GenBanka Y593846.1

  - FMDV serotype SAT 2 complete genome: GenBank JX014256.1

  - FMDV serotype ASIA 1 complete genome: GenBank HQ631363.1.

 Canine parvovirus VP1, VP2 protein, for example a sequence which can be indicated or derived in the following sequence:

  - Canine parvovirus VP1 gene for capsid protein VP1, partial cds, strain: 1887 / f / 3. GenBank: AB437434.1.

  - Canine parvovirus VP1 gene for capsid protein VP1, partial cds, strain: 1887 / M / 2. GenBank: AB437433.1.

  - Canine parvovirus VP2 gene, complete cds, strain: HNI-2-13. GenBank: AB120724.1.

  - Canine parvovirus VP2 gene, complete cds, strain: HNI-3-4. GenBank: AB120725.1.

  - Canine parvovirus VP2 gene, complete cds, strain: HNI-3-11. GenBank: AB120726.1.

  - Canine parvovirus VP2 gene, complete cds, strain: HNI-4-1. GenBank: AB120727.1.

  - Canine parvovirus VP2 gene, complete cds, strain: HNI-1-18. GenBank: AB120728.1.

  - Canine parvovirus VP2 protein (VP2) gene, complete CDS. GenBank: DQ354068.1.

  - Canine parvovirus VP2 gene, complete cds, strain: HCM-6. GenBank: AB120720.1.

  - Canine parvovirus isolate Taichung VP2 gene, complete CDS. GenBank: AY869724.1.

  - Canine parvovirus VP2 gene, complete cds, strain: HCM-8. GenBank: AB120721.1.

  - Canine parvovirus type 1 protein VP1, VP2: GenBank AB518883.1

  - Canine parvovirus type 2a VP1, VP2. GenBank: M24003.1

  - Canine parvovirus type 2b VP2: GenBank FJ005265.1

  - Canine parvovirus Type 2C VP2: GenBank FJ005248.1

 Pig parvovirus VP1, VP2 protein, such as the sequence that can be indicated or derived in the following sequence:

  - Porcine parvovirus strain 693a. GenBank: JN400519.1

  - Porcine parvovirus strain 8a. GenBank: JN400517.1

 Human parvovirus VP1, a VP2 protein, for example a sequence that can be indicated or derived in the following sequence:

  - Human parvovirus B19 VP1 complete CDS. GenBank: AF264149.1

  - Human parvovirus B19 isolate Vn115 NS1 (NS1), 7.5 kDa protein (NS1), VP1 (VP1), 9.5 kDa protein (VP1), VP2 (VP2) genes, complete CDS. GenBank: DQ357065.1

  - Human parvovirus B19 isolate FoBe VP1 (VP1), VP2 (VP2) genes, complete CDS. GenBank: AY768535.1

  - Human parvovirus B19 isolate Br543 NS1 (NS1), VP1 (VP1), VP2 (VP2) genes, complete CDS. GenBank: AY647977.1

  - Human parvovirus 4 isolate VES065CSF NS1, VP1, VP2 genes, complete cds. GenBank: HQ593532.1

  - Human parvovirus 4 isolate VES085CSF NS1 gene, partial CDS, VP1, VP2 genes, complete CDS. GenBank: HQ593531.1

  - Human parvovirus B19 strain BB-2 NS1, VP1, VP2 genes, complete CDS. GenBank: KF724387.1

 Norovirus genogroup I or II envelope proteins, such as sequences that can be indicated or derived in the following sequence:

  - Norwalk virus: GenBank Μ87661, NP056821

  - Southampton virus: GenBank L07418

  - Mexico virus: GenBank U22498

  - Seto virus: GenBank AB031013

  - Chiba virus: GenBank AB042808

  - Lordsdale virus: GenBank X86SS7

  - Snow Mountain virus: GenBank U70059

  - Hawaii virus: GenBank U07611

 Rabbit hemorrhagic virus (RHDV) VP60 protein, for example, a sequence that can be indicated or derived in the following sequence:

  - RHDV AST / 89 complete genome: GenBank: Z49271.2

  - RHDV N11 complete genome: GenBank: KM878681.1

  - RHDV CBVal16 complete genome; GenBank: KM979445.1

  SEQ ID NO .: 32

  SEQ ID NO .: 33

 Human papillomavirus (HPV) L1 protein, for example, a sequence that can be indicated or derived in the following sequence:

  - HPV 6: GenBank: JN252323.1

  - HPV 11: GenBank: JQ773411.1

  - HPV 16: GenBank DQ155283.1

  - HPV 18: GenBank FJ528600.1

 E hepatitis virus E2 protein, for example a sequence which can be indicated or derived in the following sequence:

  - Hepatitis E virus, complete genome NCBI Reference Sequence: NC_001434.1

  - Swine hepatitis E virus isolate ITFAE11 capsid protein gene. GenBank: JN861806.1

 Infectious F pancreatic virus VP1, VP2, VP3 proteins, for example sequences which can be indicated or derived in the following sequence:

  - Infectious bursal disease virus VP1 (VP1) gene, complete CDS. GenBank: AY099457.1

  - Infectious bursal disease virus isolate PT VP1 gene, complete CDS. GenBank: DQ679814.1

  - Infectious bursal disease virus isolate OE / G2 VP1 gene, complete CDS. GenBank: DQ679813.1

  - Infectious bursal disease virus isolate OA / G1 VP1 gene, complete CDS. GenBank: DQ679812.1

  - Infectious bursal disease virus isolate HOL VP1 gene, complete CDS. GenBank: DQ679811.1

  - Infectious bursal disease virus strain TL2004 VP1 gene, complete CDS. GenBank: DQ 118374.1

  - Infectious bursal disease virus isolate CA-K785 VP1 gene, complete CDS. GenBank: JF907705.1

  - Infectious bursal disease virus isolate D495 VP1 gene, complete CDS. GenBank: JF907704.1

  - Infectious bursal disease virus strain A-BH83 VP1 mRNA, complete cds. GenBank: EU544149.1

  - Infectious bursal disease virus strain Cro-Pa / 98 VP1 gene, complete CDS. GenBank: EU184690.1

  - Infectious bursal disease virus VP2 mRNA, complete cds. GenBank: AY321509.1

  - Infectious bursal disease virus VP2, VP3, VP4 genes, complete CDS. GenBank: M97346.1

  - Infectious bursal disease virus VP2 gene, complete CDS. GenBank: AF508177.1

 A calicivirus coat protein, for example, a sequence that can be indicated or derived in the following sequence:

  - Feline calicivirus capsid protein gene, complete cds. GenBank: L09719.1

  - Feline calicivirus capsid protein gene, complete cds. GenBank: L09718.1

  - Human calicivirus HU / NLV / Wortley / 90 / UK RNA for capsid protein (ORF2), strain HU / NLV / Wortley / 90 / UK. GenBank: AJ277618.1

  - Human calicivirus HU / NLV / Thistlehall / 90 / UK RNA for capsid protein (ORF2), strain HU / NLV / Thistlehall / 90 / UK. GenBank: AJ277621.1

  - Human calicivirus HU / NLV / Valetta / 95 / Malta RNA for capsid protein (ORF2), strain HU / NLV / Valetta / 95 / Malta. GenBank: AJ277616.1

  - Human calicivirus NLV / Stav / 95 / N or capsid protein gene, complete CDS. GenBank: AF145709.1

  - Bovine enteric calicivirus strain Bo / CV500-OH / 2002 / US capsid protein gene, complete CDS. GenBank: AYS491SS.1

  - Human calicivirus NLV / Mora / 97 / SE capsid protein gene, complete CDS. GenBank: AY081134.1

  - Human calicivirus NLV / Potsdam 196/2000 / DE capsid protein gene, complete cds. GenBank: AF439267.1

  - Human calicivirus NLV / 1581-01 / SWE capsid protein gene, complete CDS. GenBank: AY247442.1

  - Human calicivirus Hu / NLV / GII / MD134-10 / 1987 / US capsid protein gene, complete CDS. GenBank: AY030313.1

 An astrovirus ORF2-encoded protein, such as a sequence that can be indicated or derived in the following sequence:

  - Porcine astrovirus 4 ORF1b gene, partial CDS; ORF2 gene, complete CDS. GenBank: JX684071.1

  - Astrovirus MLB1 HK05, complete genome. NCBI Reference Sequence: NC 014320.1

  - Astrovirus wild boar / WBAstV-1/2011 / HUN, complete genome. NCBI Reference Sequence: NC_016896.1

  - Human astrovirus BF34, complete genome. NCBI Reference Sequence: NC 024472.1

  - Astrovirus MLB1 strain Hu / ITA / 2007 / PR326 / MLB1 RNA-dependent RNA polymerase (ORF1b) gene, partial cds, capsid protein (ORF2) gene, complete cds. GenBank: KF417713.1

  - Human astrovirus 5 strain Hu / Budapest / HUN5186 / 2012 / HUN nonstructural protein (ORF1a), nonstructural protein (ORF1b) genes, partial cds, capsid protein (ORF2) gene, complete cds. GenBank: KF157967.1.

  - Human astrovirus 1 isolate Shanghai capsid protein (ORF2) gene, complete cds. GenBank: FJ792842.1

  - Human astrovirus type 8 orf2 gene for capsid protein. GenBank: Z66541.1

 Influenza virus HA, NA, M1 protein, for example sequences which can be indicated or derived in the following sequence:

  SEQ ID NO .: 30

  - Influenzae virus (A / duck / Chiba / 25-51-14 / 2013 (H7N1)) HA gene for hemagglutinin, complete cds. GenBank: AB813060.1

  Synthetic construct hemagglutinin (HA) mRNA, complete cds. GenBank: DQ868374.1

  - Influenza virus A (A / swine / Shandong / 2/03 (H5N1)) hemagglutinin (HA) gene, complete CDS. GenBank: AY646424.1

  - cDNA encoding HA of influenza type A. GenBank: E01133.1

  - Influenza A virus (A / swine / Korea / S452 / 2004 {H9N2)) NA gene, complete CDS. GenBank: AY790307.1

  - Influenza A virus (A / Thailand / 2 (SP-33) / 2004 {H5N1)) neuraminidase (NA) gene, complete CDS. GenBank: AYSSS 152.3

  - Influenza virus (A / swine / Binh Doung / 02_ 16/2010 (H1N2)) NA gene for neuraminidase, complete cds. GenBank: AB628082.1

  - Influenza A virus (A / chicken / Jalgaon / 8824/2006 (H5N1)) neuraminidase (NA) gene, complete CDS. GenBank: DQ887063.1

  - Influenza A virus SC35M M2, M1 genes, complete CDS. GenBank: DQ266100.1

  - Influenza virus type / Leningrad / 134/47/57 (H2N2) M1, M2 RNA, complete CDS's. GenBank: M81582.1

  - Influenza A virus SC35M M2, M1 genes, complete CDS. GenBank: DQ266100.1

  - Influenza A virus (A / Tochigi / 2/2010 (H1N1)) M2, M1 genes for matrix protein 2, matrix protein 1, complete cds. GenBank: AB 704481.1

 Hepatitis B virus core antigens and surface antigens, for example sequences that can be indicated or derived in the following sequence:

  - Hepatitis B virus strain HBV248 precore protein, core protein genes, complete cds. GenBank: KP857118.1

  - Hepatitis B virus strain HBV401 precore protein, core protein genes, complete CDS. GenBank: KP857113.1

  - Hepatitis B virus strain HBV403 precore protein, core protein genes, complete cds. GenBank: KP857068.1

- surface antigen, partial cds, isolate: B0503327 (PTK). GenBank: AB466596.1

The step (2) of spraying or immersing the recombinant baculovirus in the flounder of the present invention and inoculating the recombinant protein and expressing the recombinant protein can be carried out in the order of Pyralis farinalis, Paralipsa gularis, Plodia interpunctella Selected from the group consisting of Aglossa dimidiatus, Corcyra cephalonica, Ephestia kuehniella, Ephestia elutella, and Cadra cautella. After packaging in a sterilized container belonging to one of the Pyralid moths to which the food is added and having a chick embryo and a wax layer removed from the egg shell and disinfected and having a ventilation structure, Preferably stored in a Mediterranean flour moth species, packed in a sterile container having a ventilation structure with the chick embryo and wax layer removed from egg shells, It supplies stored moth eggs.

Then, the recombinant baculovirus originating from Autographa californica nuclear polyhedrosis virus (AcMNPV) is sprayed onto the above-mentioned birds or preferably the above-mentioned birds are immersed in a liquid containing recombinant baculovirus derived from AcMNPV Lt; RTI ID = 0.0 > AcMNPV < / RTI > Also incubated with recombinant baculovirus from AcMNPV for a time sufficient to express at least one recombinant protein.

Finally, the recombinant protein is cultured for a time sufficient for expression of the recombinant protein to recover the frog containing the at least one recombinant protein, and then stored frozen at -20 캜. Under the above -20 ° C freezing condition, the moths can be stored for up to about one year, and thus it is possible to supply them to the whole world through the refrigeration transportation process using airplanes and ships.

The step (3) of harvesting and purifying the recombinant protein in the present invention of the present invention to produce a product comprises the step (3), which comprises at least one recombinant protein and which is frozen and stored at -20 캜. And crushes the frozen frozen egg containing the at least one recombinant protein. Finally, the at least one recombinant protein is isolated and purified from the liquid in which the flounder is pulverized to produce a final recombinant protein product.

The present invention may be embodied in many other specific forms without departing from the spirit or essential characteristics of the invention.

1: The process of removing the chorion and wax layer of egg shell egg shell
2: Inoculation of recombinant baculovirus into spikelets and inoculation and expression of recombinant proteins
3: Producing products by harvesting and purifying recombinant proteins from the moths

Claims (4)

Pyralis farinalis, Paralipsa gularis, Plodia interpunctella, Aglossa dimidiatus, Corcyra cephalonica, Pyralid moths, which affect the stored food, , Ephestia kuehniella, Ephestia elutella, and Cadra cautella, belonging to one species selected from the group consisting of Autograpa californica nuclear polyhedrosis virus Wherein the recombinant baculovirus comprises at least one of recombinant baculovirus and bacmids comprising a nucleic acid sequence encoding a recombinant protein derived from AcMNPV.
The method according to claim 1,
The recombinant protein may be selected from the group consisting of a subunit monomeric vaccine, a subunit multimeric vaccine, a virus like particle, a therapeutic protein, an antibody, an enzyme (GFP), a fluorescent protein (GFP), an enzyme, a cytokine, a blood clotting factor, an anticoagulant, a receptor, a hormone, a diagnostic protein reagent, protein), characterized in that the recombinant protein is not an endogenously produced protein by a pupa.
Pyralis farinalis, Paralipsa gularis, Plodia interpunctella, Aglossa dimidiatus, Corcyra cephalonica, and Mediterranean flour, which are food additives, A method for producing a chrysanthemum of egg shell belonging to any one species selected from the group consisting of Ephestia kuehniella, Ephestia elutella, and Cadra cautella, As a result,
(a) providing a protozoan egg surrounded by an egg shell having a multilayer structure of a vitelline membrane, a waxy layer, and a chorionic layer;
(b) treating chick embryo surrounded by egg shells with a non-toxic chemical, sodium hypochlorite, to remove chorion;
(c) treating the flounder of step (b) with a non-toxic composition comprising D-limonene, Cocamide DEA, and ethoxylated alcohol, ; And
(d) recovering the protozoan egg from which the chick embryo and wax layer of egg shell are removed and the surface is disinfected.
A method for producing at least one recombinant protein,
(a) Pyralis farinalis, Paralipsa gularis, Plodia interpunctella, Aglossa dimidiatus, Corcyra cephalonica, Ephestia kuehniella, , Ephestia elutella, and Cadra cautella, and the surface of which is disinfected with a chick embryo of the egg shell;
(b) a step of spraying the recombinant beculovirus originating from Autographa californica nuclear polyhedrosis virus (AcMNPV) to the protozoan egg of step (a), or a step (a) of adding a recombinant baculovirus derived from AcMNPV, Inoculation by immersing the mosquito eggs of the mosquito;
(c) culturing the inoculated protozoan eggs of step (b) for a time sufficient to allow expression of at least one recombinant protein;
(d) recovering the protozoan eggs comprising at least one recombinant protein;
(e) harvesting at least one recombinant protein; And
(f) purifying at least one recombinant protein. < RTI ID = 0.0 > 8. < / RTI >

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